2018 Fiscal Year Annual Research Report
Cross-organ HSC trafficking during definitive hematopoiesis formation
| Project/Area Number |
18J20690
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| Research Institution | Kumamoto University |
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Principal Investigator |
瀬崎 真衣子 熊本大学, 医学教育部, 特別研究員(DC1)
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| Project Period (FY) |
2018-04-25 – 2021-03-31
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| Keywords | 3D whole-organ imaging / hematopoietic switching / deep-learning analysis / EC remodeling |
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| Outline of Annual Research Achievements |
Till now, hematopoietic stem cell (HSC) trafficking has been characterized via flow cytometry spatially i.e., liver, spleen, bone marrow (BM) and temporally (E14.5 to P14) in terms of HSC (LT- and ST-HSCs) and MPP (2 and 3/4) absolute number. Results revealed a peak in HSC expansion in the liver around P0, followed by a gradual “hematopoietic switching” to the BM thereafter. Together with cell cycle and apoptosis assays, data is currently being used to devise a mathematical model to delineate cross-organ mobilization of HSCs and each organ’s contribution to the overall HSC pool, as well as HSC character (self-renewal vs differentiation).
3D whole-organ imaging has been adapted for fetal~neonatal BM samples and applied for imaging of Hlf-tdTomato (a novel HSPC tracer mouse line) femurs together with vascular markers (endomucin and CD31) or along with several stromal mouse lines (i.e., LepR-cre) that may play a role in HSC transit. Quantification using Imaris showed sub-populations of all stromal lines to be in direct contact with potential HSCs within the BM. Femur bones of P2 and P5 mice are currently being processed for imaging of the outside surface.
Femurs imaged at E18.5 suggest an initial colonization of c-Kit+tdTomato+ cells on the outside surface of the bone near the vascular network of the periosteum. These cells (HSCout) were physically separated from c-Kit+tdTomato+ cells inside (HSCin) and single-cell sorted for RNA sequencing. Top ~100 genes are currently being examined for each cluster to reveal those possibly playing a role in HSC transit within the BM.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Overall, data obtained until now is mostly phenotypic and functional/quantitative analysis is underway. However, with regards to the progress of this project, all necessary techniques and tools (i.e., 3D whole-organ imaging, stromal mouse lines, single-cell RNA sequencing) have been established, national and international collaborations actively commenced (University of Tokyo, University of Zurich, University of Cambridge) and preliminary data amply acquired and partly analyzed. Progression of this project is according to plan. Regarding RNA sequencing, preliminary data analysis of HSCs single-cell sorted at P2 revealed distinct clusters. The list of genes that are differentially expressed have proven unique and highly promising. Much progress is to be expected of this year.
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| Strategy for Future Research Activity |
Acquired imaging data of P2 femurs will be analyzed via a deep-learning based algorithm currently being tailored by our collaborators for automated high-throughput readout of vascular and stromal cell populations. The most likely candidate(s) involved in HSC transit within the BM will be determined and from there, RNA sequencing of the “niche-side” will be considered. From the differentially expressed gene list of P2 HSCin and HSCout clusters, several will be selected and validated. Through silencing of target genes in HSCs and transplantation experiments, the necessity of each for HSC trafficking and HSC transit within the BM will be checked. In parallel, tracing experiments and HSC clonality will be investigated using two strategies: 1. barcoding of individual HSCs using the sleeping beauty transposon system or 2. flow cytometry and imaging of confetti mice crossed with Hlf-creERT2 (tamoxifen inducible) for single-cell labelling and tracing of HSC progeny spatiotemporally.
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